Mutated immunoglobulin-binding polypeptides

A technology that combines peptides and staphylococcal proteins, applied in the direction of immunoglobulin, carrier-bound/immobilized peptides, peptides, etc., can solve the problems of difficult to achieve cleaning, unacceptable loss of ability, etc., and achieve the effect of highly selective binding

Active Publication Date: 2018-12-21
CYTIVA BIOPROCESS R&D AB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, currently available mutants are still sensitive to alkaline pH, and the NaOH concentration during cleaning is usually limited to 0.1 M, which means that complete cleaning is difficult to achieve.
Higher NaOH concentrations (which would improve cleaning) lead to unacceptable loss of capacity

Method used

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  • Mutated immunoglobulin-binding polypeptides
  • Mutated immunoglobulin-binding polypeptides
  • Mutated immunoglobulin-binding polypeptides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0293] The purified monomeric ligands listed in Table 1 (for SEQ ID NOs 8-16, 23-28 and 36-48, also containing the AQGT leader sequence at the N-terminus and the cysteine ​​at the C-terminus), Using GE Healthcare's Amine Coupling Kit (for carbodiimide coupling of amines on carboxymethyl groups on a chip) sufficient to give approx. Amounts of signal intensity ranging from 200-1500 RU were immobilized on Biacore CM5 sensor chips (GE Healthcare, Sweden). To follow the IgG binding capacity of the immobilized surface, 1 mg / ml human polyclonal IgG (Gammanorm) was flowed over the chip and the signal intensity (proportional to the amount bound) was recorded. The surface was then cleaned in place (CIP), ie rinsed with 500 mM NaOH for 10 minutes at room temperature (22 + / - 2°C). 96-100 cycles were repeated and the immobilized ligand alkali stability was followed after each cycle by remaining IgG binding capacity (signal intensity). The results are shown in Table 1 and show that at lea...

Embodiment 2

[0300]Purified dimer, tetramer and hexameric ligands listed in Table 2 using GE Healthcare's Amine Coupling Kit (for carbodiimide coupling of amines on carboxymethyl groups on the chip) , was immobilized on a Biacore CM5 sensor chip (GE Healthcare, Sweden) in an amount sufficient to give a signal intensity of approximately 200-1500 RU in a Biacore instrument (GE Healthcare, Sweden). To follow the IgG binding capacity of the immobilized surface, 1 mg / ml human polyclonal IgG (Gammanorm) was flowed over the chip and the signal intensity (proportional to the amount bound) was recorded. The surface was then cleaned in place (CIP), ie rinsed with 500 mM NaOH for 10 minutes at room temperature (22 + / - 2°C). 300 cycles were repeated and the immobilized ligand alkali stability was followed after each cycle by remaining IgG binding capacity (signal intensity). The results are shown in Table 2 and figure 2 and showed that at least the ligands Zvar(Q9A,N11E,N43A)4, Zvar(Q9A,N11E,N28A,N...

Embodiment 3

[0305] Example 2 was repeated with 100 CIP cycles of 3 ligands using 1 M NaOH instead of 500 mM in Example 2. The results are shown in Table 3 and show that all 3 ligands also have improved alkaline stability in 1M NaOH compared to the parental structure Zvar4 used as reference.

[0306] Table 3. Tetrameric ligands evaluated with Biacore (1M NaOH).

[0307] Ligand

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PUM

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Abstract

An Fc-binding polypeptide of improved alkali stability, comprising a mutant of aparental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 22, SEQ ID NO 51 or SEQ ID NO 52, wherein at least the asparagine or serine residue at the position corresponding to position 11 in SEQID NO: 4-7 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine.

Description

technical field [0001] The present invention relates to the field of affinity chromatography, and more particularly to mutated immunoglobulin-binding domains of protein A, which can be used in affinity chromatography of immunoglobulins. The invention also relates to multimers of said mutated domains and to separation matrices containing mutated domains or multimers. Background of the invention [0002] Immunoglobulins represent the most common biopharmaceutical product produced or developed worldwide. The high commercial demand and consequent value for this particular therapeutic market has led to a focus on pharmaceutical companies to maximize the productivity of their respective mAb production processes while controlling the associated costs. [0003] Affinity chromatography is used in most cases as one of the key steps in the purification of these immunoglobulin molecules, eg monoclonal or polyclonal antibodies. One class of affinity reagents of particular interest are ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/31G01N33/68
CPCB01J20/267B01J20/285B01J20/286B01J20/3274B01J2220/52C07K1/22C07K14/31C07K16/00C07K17/10B01D15/3809B01J20/28016B01J20/28078B01J20/3212C07K16/065B01J20/24C07K16/1271
Inventor G.J.罗德里戈T.布约克曼M.安德
Owner CYTIVA BIOPROCESS R&D AB
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